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Polygalacturonic acid/endo-polygalacturonase system: a kinetic study in batch reactors.

The enzymatic depolymerization of the pectic substance polygalacturonic acid (PGA) is studied in batch reactor. The number-average molecular weight of native substrate is estimated, using a simple and quick technique, to be approximately 11.1 kDa, the polymeric chains consisting on average of 63 galacturonic acid units. The effect of enzyme concentration was studied varying biocatalyst loading from 6 to 242 mg/L. The experiments were repeated at substrate concentrations ranging from 0.5 to 5 g/L. Data obtained at both short reaction time (20 min) and prolonged enzyme action (up to 350 min) are correlated using different kinetic equations, and the parameter values are discussed.

Bioreactors↗

Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures.

Late exponential phase hairy root cultures of Catharanthus roseus were elicited with pectinase and jasmonic acid. The effects of elicitor concentration and exposure time on growth and levels of several compounds in the indole alkaloid biosynthetic pathway were monitored. Pectinase decreased the fresh weight to dry weight ratio of the roots, while addition of jasmonic acid had no significant effect. Selective effects on indole alkaloid yields were observed upon addition of elicitors. An increase of 150% in tabersonine specific yield was observed upon addition of 72 units of pectinase. Transient studies at the same level demonstrated possible catabolism as serpentine, tabersonine, and lochnericine levels decreased immediately after elicitation. The levels of these compounds recovered back to control levels or were higher than the control levels after some time. Jasmonic acid was found to be a unique elicitor leading to an enhancement in flux to several branches in the indole alkaloid pathway. Jasmonic acid addition caused an increase in the specific yields of ajmalicine (80%), serpentine (60%), lochnericine (150%), and hörhammericine (500%) in dosage studies. Tabersonine, the likely precursor of lochnericine and hörhammericine, decreased at lower levels of jasmonic acid and then increased with increasing jasmonic acid concentration. Transient studies showed that lochnericine and tabersonine levels go through a maxima, then decrease back to control levels and reduce below control levels, respectively. The yields of ajmalicine, serpentine, and hörhammericine increased continuously after the addition of jasmonic acid. The methods described in this article could generally be used in devising strategies for enhancement in productivity of secondary metabolites and for probing and studying the complex secondary metabolite pathways in plant tissue cultures.

Alkaloids↗

Changes in essential oil during enzyme-assisted ensiling of lemongrass (Cymbopogon citratus Stapf.) and lemon eucalyptus (Eucalyptus citriodora Hook).

Changes in essential oil during ensiling of lemongrass and lemon eucalyptus were studied. Wilted lemongrass and eucalyptus leaves were ensiled in 0.25-L anaerobic jars. Samples consisted of a control (no additives) and a treated sample (0.5% glucose and lactic acid bacteria and 1% cellulase plus 1% hemicellulase plus pectinase). Three jars per treatment were sampled on days 2, 6, 10, and 36 for analysis of essential oil. Essential oil was obtained by extraction and by hydrodistillation. Extraction efficacy of essential oil from the lemongrass was improved by the enzyme treatment, but it was much lower than the amount obtained by distillation. The major components of the essential oil were neral and geranial. In the eucalyptus, total essential oils obtained by distillation decreased during ensiling, and the amount was similar to the amount obtained by extraction. Citronellal, which was the major component of the essential oil in the fresh eucalyptus leaves, decreased, whereas isopulegol and 3,8-terpinolhydrate increased during ensiling.

Anti-Bacterial Agents↗

Effects of commercial pectolytic and cellulolytic enzyme preparations on the apple cell wall.

The action of three different commercial enzyme combinations on apple cell wall material has been examined in a model system under conditions of mash and pomace treatment by using an alcohol-insoluble substance prepared from apples. A part of the total dietary fiber, for example, galacturonan (pectin), appeared in the soluble fraction after enzymatic mash treatment. The soluble fraction increased intensely during pomace treatment. Furthermore, enzyme actions caused a change in the water-binding capacity of residues as well as changes in the monosaccharide composition and in the molecular weight distribution of saccharides in filtrates (soluble parts). The extent of decomposition of cell wall material and the increase of soluble oligomeric and/or polymeric dietary fiber components are caused by both the composition (pectinases, cellulases, and hemicellulases) and the activities of the enzyme preparations. The model experiments allow an insight into the reactions occurring during enzyme action on the plant cell wall, for example, during apple juice production using pectolytic and cellulolytic enzyme preparations.

Cell Wall↗

Soybean (Glycine max) cell wall composition and availability to feed enzymes.

Defatted untoasted soybean cotyledons and hulls were fractionated as water solutes (WSc and WSh) and water unextractable (WUc and WUh). Further fractionation of WUc through deproteinization yielded the isolation of a water unextractable solid (WUS) fraction that was mainly composed (molar percent) of galactose (28.1%), glucose (27.8%), arabinose (13.3%), and uronic acids (17.6%), which accounted for 76% of the water insoluble polysaccharides in soybean cotyledons (WUc). The cell wall (WUS) was sequentially fractionated with chelating agents (chelating agent soluble solids, ChSS) and a gradient of agents (dilute alkali, DASS; 1 M alkali, 1MASS; and 4M alkali, 4MASS), which gave a final cellulosic residue. The ChSS and DASS extracts were characterized as pectin-rich fractions, whereas 1MASS and 4MASS were hemicellulose- and cellulose-rich fractions. Incubation in vitro of the WUc fraction with pectinase, cellulase, and xylanase resulted in the release of low amounts (not more than 5% bound basis) of monosaccharides, mostly uronic acids, xylose, and arabinose. Protein extraction hardly increased this release after enzymatic incubation (<7%). However, progressive fractionation of the cell wall matrix markedly increased the release of monosaccharides from pectin- (ChSS and DASS) and hemicellulose-rich fractions (1MASS and 4MASS). Significant degradation of cellulose (up to 20%) was achieved only after complete protein, pectin, and hemicellulose extraction.

Arabinose↗

Structural characterization of oligosaccharides and polysaccharides from apple juices produced by enzymatic pomace liquefaction.

Eight apple pomace liquefaction juices were produced to characterize soluble cell wall material released by the action of pectolytic and cellulolytic enzyme preparations. Very high colloid values from 9.7 to 19.6 g/L were recovered from the juices by ethanol precipitation. The crude polysaccharides consisted mainly of galacturonic acid (49-64 mol %), arabinose (14-23 mol %), galactose (6-15 mol %), and minor amounts of rhamnose, xylose, and glucose. Separation of the polysaccharides by anion-exchange chromatography yielded one neutral, one slightly acidic, and one acidic polymer accounting for 60% of total colloids. Preparative size exclusion chromatography of the acidic fractions resulted in four polymers of different molecular weights and different sugar compositions. Among them, high molecular weight arabinans and rhamnogalacturonans as well as oligomeric fractions consisting of only galacturonic acid could be found. Linkage studies were performed on neutral fractions from anion-exchange chromatography and size exclusion chromatography. They revealed highly branched arabinans, xyloglucans, and mainly type I arabinogalactans.

Beverages↗

Chemical composition and enzymatic degradability of xylem and nonxylem walls isolated from alfalfa internodes.

During plant maturation, degradability of alfalfa (Medicago sativa L.) stems declines due to accumulation of highly lignified xylary tissue. Xylem and nonxylem tissues dissected from lower alfalfa internodes were analyzed for cell wall constituents and degradability. Cell walls comprised 740 mg g(-1) of xylem and 533 mg g(-1) of nonxylem tissues. Xylem tissues contributed about 60% of the cell wall mass in internodes. Xylem walls contained 28% lignin, 4% pectin, 29% hemicellulose, and 39% cellulose as compared to 15% lignin, 25% pectin, 30% hemicellulose, and 30% cellulose in nonxylem walls. Fungal enzymes hydrolyzed 22 and 73% of the structural carbohydrates in xylem and nonxylem walls, respectively. In both cell wall fractions, the release of xylose was 56-90% lower than that of other sugars, indicating that lignin preferentially restricted xylan degradation in secondary walls and xyloglucan degradation in primary walls. Elucidation of lignin-xylose interactions may reveal strategies for improving fiber degradability of alfalfa.

Cell Wall↗

Multivariate evaluation of changes induced in red wine characteristics by the use of extracting agents.

The aim of the present work was to study the effect of both vintage and the use of four types of pectinolytic enzymatic preparations on the quality of red wine using factor analysis. We also studied the evolution of the factors obtained along two years of storage of the wine. The results obtained from the factor analysis revealed that the addition of pectinolytic enzymes enriched the phenolic composition and elicited an improvement in the visual aspect of the wine, which persisted during the storage period considered.

Analysis of Variance↗

Enzyme-mediated solvent extraction of carotenoids from marigold flower (Tagetes erecta).

Marigold flowers are the most important source of carotenoids for application in the food industry. However, the extraction gives almost 50% losses of the carotenoids depending on conditions for silaging, drying, and solvent extraction. In the past decades, macerating enzymes have been successfully applied to improve the extraction yield of valued compounds from natural products. In this work, an alternative extraction process for carotenoids is proposed, consisting of a simultaneous enzymatic treatment and solvent extraction. The proposed process employs milled fresh flowers directly as raw material, eliminating the inefficient silage and drying operations as well as the generation of hard to deal with aqueous effluents present in traditional processes. The process developed was tested at the 80 L scale, where under optimal conditions a carotenoid recovery yield of 97% was obtained.

Asteraceae↗

Considerations on endopolygalacturonase activity and determination of comparison ratios with emphasis on the influence of the degree of substrate esterification.

A study on the determination and standardization of endopolygalacturonase (EPG) activity is reported, with emphasis on the influence of the degree of substrate esterification using pure yeast EPG. Differences in the results, depending on how the EPG activity unit was defined, are described and discussed. From a theoretical analysis of the expressions established, a general equation for expressing EPG activity in standard international units was obtained, together with the proportional coefficient for each of the substrates studied. It was observed that for a wide range of enzyme concentrations good linear correlations were obtained. Analysis of the comparison ratio (CR) values calculated revealed that these do not differ significantly, except for low-methoxyl apple pectin, confirming the validity of the general expression obtained for pectins with different degrees of esterification. The anomalous CR value found for low-methoxyl (LM) apple pectin is discussed.

Esterification↗

Investigation of bound aroma constituents of yellow-fleshed nectarines (Prunus persica L. Cv. Springbright). changes in bound aroma profile during maturation.

Glycosidically bound volatile constituents of yellow-fleshed clingstone nectarines (cv. Springbright) were identified and quantified at three stages of maturity. Glycoconjugates were isolated by LC on a C(18) reversed phase column with methanol elution followed by hydrolysis with a commercial pectinase enzyme. Forty-five bound aglycons were identified for the first time in yellow-fleshed nectarine. Thirty were terpene derivatives, and the most abundant ones were (E)- and (Z)-furan linalool oxides, linalool, alpha-terpineol, (E)-pyran linalool oxide, 3,7-dimethylocta-1,5-diene-3,7-diol, linalool hydrate, 8-hydroxy-6,7-dihydrolinalool, (E)- and (Z)-8-hydroxylinalools, and (E)- and (Z)-8-hydroxygeraniols. The group of C(13) norisoprenoids included 3-hydroxy-beta-damascone, 3-hydroxy-7,8-dihydro-beta-ionone, 3-oxo-alpha-ionol, 3-hydroxy-7,8-dihydro-beta-ionol, 3-hydroxy-beta-ionone, 3-oxo-7,8-dihydro-alpha-ionol, 3-hydroxy-5,6-epoxy-beta-ionone, 3-oxo-retro-alpha-ionol (isomers I and II), 3-hydroxy-7,8-dehydro-beta-ionol, 4,5-dihydrovomifoliol, and vomifoliol. Generally, levels of bound compounds, in particular monoterpenols and C(13) norisoprenoids, increased significantly with maturation. delta-Decalactone was the only lactone found in the enzymatic hydrolysate of yellow-fleshed nectarine, but its level was much lower than that of its free form.

Chromatography, Gas↗

Enzymatic high digestion of soybean milk residue (okara).

The objective of this study was to digest okara in high yield by food-processing enzymes. Autoclaving of okara was effective in increasing cellulase digestion for the primary cell wall, and the digestion was accelerated by the formation of single cells by stirring. Most of the residual okara after autoclaving and cellulase digestion was found to be the secondary cell walls compared with the cellulase-treated soybean single cells. The secondary cell wall was found to be composed of galacturonic acid, neutral sugars, and protein and was considered to be a complex of these compositions. Many cellulolytic and proteolytic enzymes could not digest the secondary cell wall; however, it was found that two pectinases could digest the secondary cell wall. A series of digestions resulted in yields of 83-85% from the raw okara, and the final residues were identified as oil body complexes in the soybean cells and fiber-like organ between the cells.

Cell Wall↗

Effect of pectolytic enzyme preparations on the phenolic composition and antioxidant activity of asparagus juice.

Commercial pectolytic enzymes were investigated for their influence on phenolics and antioxidant activities of asparagus juice. The antioxidant activity of asparagus juice was analyzed according to 2,2'-diphenyl-l-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) methods. The enzymes, with the exception of pectinase from Rhizopus sp., contained rutinase, which hydrolyzed rutin to quercetin. Asparagus juice treated with Viscozyme had the highest quercetin content without exhibiting a significant increase in the antioxidant activity. For a pectinase from Aspergillus niger, the antioxidant activity of asparagus juice was markedly reduced. Caution should be paid in the selection of pectolytic enzyme preparations for production of antioxidant activity-rich juice.

Antioxidants↗

Study of the role of the carbohydrate and protein moieties of soy soluble polysaccharides in their emulsifying properties.

The objective of this work was to investigate the role played by the protein fraction of soy soluble polysaccharide (SSPS) during its adsorption at oil/water interfaces. SSPS was separated in a high (HMF; 310 kDa) and low (LMF; 20 kDa) molecular weight fraction by gel filtration. SSPS/HMF consisted of 91.6% carbohydrate and 2.2% protein and showed better emulsifying properties than those of the whole SSPS, whereas SSPS/LMF seemed to affect negatively the adsorption behavior of SSPS. SDS-PAGE of the protein fraction obtained from SSPS/HMF showed a molecular mass of 50 kDa, was composed predominantly of proline (23.1%) and glutamic acid (15.2%), and still contained 8.8% of neutral sugar and 5.3% of uronic acid. Results indicated that not all of the protein material present in SSPS contributes to SSPS functionality but that only the material associated with HMF aids in the adsorption of SSPS onto oil/water interfaces.

Adsorption↗

Combined enzymatic and high-pressure processing affect cell wall polysaccharides in berries.

The effect of high-pressure processing (HPP) on cell wall polysaccharides in berries was investigated. HPP decreased the degree of methyl esterification (DM), probably by activation of pectin methyl esterase (PME), and improved the extractability of pectins. When commercial enzyme mixtures were added to mashed berries, a synergistic effect was observed between treatment with commercial enzymes and HPP. Compared to treatment at atmospheric pressure, pectic polysaccharides were degraded to a larger extent when HPP was used. In contrast, hemicelluloses were hardly affected by the added enzymes when HPP was included, although they were degraded during similar treatment at atmospheric pressure. Additionally, the activity of rhamnose-releasing enzymes present in minor quantities might be enhanced after HPP, resulting in a decrease of rhamnose in the polymeric cell wall material. These results exploring the effect of HPP at representative conditions clearly point out the potential of HPP for polysaccharide modification.

Acetylation↗

Beet sugar syrup and molasses as low-cost feedstock for the enzymatic production of fructo-oligosaccharides.

Sugar syrup and molasses from beet processing containing 620 and 570 mg/mL sucrose, respectively, were assayed as low-cost and available substrates for the enzymatic synthesis of fructo-oligosaccharides (FOSs). A commercial pectinase (Pectinex Ultra SP-L, from Aspergillus aculeatus) characterized by the presence of a transfructosylating activity was used as a biocatalyst. The FOS production increased when lowering the initial pH value of syrup (7.5) and molasses (8.9) to 5.5. Sugar syrup and molasses were diluted in order to reduce substrate viscosity; interestingly, the percentage of FOS with regards to total sugars remained almost constant, which indicated a high transferase-to-hydrolase ratio for this enzyme. Kinetics of FOS production was analyzed. Using approximately 10 U transfructosylating activity per g sucrose, the FOS concentration reached a maximum of 388 mg/mL after 30 h using syrup and 235 mg/mL in 65 h with molasses. These values corresponded to approximately 56 and 49% (w/w), respectively, of the total amount of carbohydrates in the mixture. The enzyme was also covalently immobilized on an epoxy-activated polymethacrylate-based polymer (Sepabeads EC-EP5). We found that immobilized Pectinex Ultra SP-L can be efficiently applied to the synthesis of FOS using syrup and molasses as substrates.

Animal Feed↗

Protease-assisted clarification of black currant juice: synergy with other clarifying agents and effects on the phenol content.

Conventional clarification with gelatin and silica sol removes a considerable amount of antioxidant phenolics from berry juices. This study examined the clarification and haze-diminishing effects of alternative clarification strategies on black currant juice including centrifugation and addition of acidic protease and pectinolytic enzyme preparations and gallic acid. Centrifugation of freshly pressed juice (10,000 g for 15 min) resulted in a approximately 95% reduction of immediate turbidity and had a decreasing effect on haze development in the juice during cold storage without significantly compromising the total phenols levels. The extent of clarification and haze diminishment varied after individual treatments with five different acidic proteases, but one of the protease preparations, Enzeco, derived from Aspergillus niger, consistently tended to perform best. The individual and interactive effects on juice turbidity, total phenols, and total anthocyanin contents of clarification treatments involving the use of two selected acid proteases (Enzeco and Novozyme 89L), a pectinase (Pectinex BE 3-L), and gallic acid were evaluated in a full factorial 2(4) experimental design. Haze development during cold storage decreased when gallic acid or any of the enzyme preparations were employed individually, but negative interaction effects resulted when the pectinase was employed in combination with any of the proteases. After 28 storage days at 2 degrees C, the lowest levels of haze formation were achieved when the Enzeco protease preparation, added at 0.025 g/L, was added with 0.050 g/L of gallic acid and allowed to react in the juice for 90 min at 50 degrees C. The corresponding anthocyanin reduction was approximately 12% (compared to approximately 30% with gelatin silica sol treatment). The data support the hypothesis that phenol-protein interactions are involved in juice turbidity development during cold storage of berry juices and demonstrate that precentrifugation and protease-assisted clarification show promise as an alternative, phenolics-retaining clarification strategy in black currant juice processing.

Anthocyanins↗

Enzymatic hydrolysis of flavonoids and pectic oligosaccharides from bergamot (Citrus bergamia Risso) peel.

Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were used to evaluate the solubilization of carbohydrates and low molecular weight flavonoids from bergamot peel, a major byproduct of the essential oil industry. The enzymes were characterized for main-chain and side-chain polysaccharide hydrolyzing activities and also against pure samples of various flavonoids previously identified in bergamot peel to determine various glycosidase activities. The addition of Pectinase 62L or 690L alone, or the combination of Pectinase 62L and Cellulase CO13P, was capable of solubilizing between 70 and 80% of the bergamot peel, and up to 90% of the flavonoid glycosides present were cleaved to their aglycones. Cellulase CO13P alone solubilized 62% of the peel but had no deglycosylating effect on the flavonoid glycosides. Over a 24-h time course, a rapid release of cell wall carbohydrates was observed after treatment with Pectinase 62L, with a concurrent gradual hydrolysis of the flavonoid glycosides. Size-exclusion chromatography of the solubilized extract showed that after 24-h incubation, the majority of the solubilized carbohydrates were present as monosaccharides with a smaller proportion of oligosaccharides.

Cellulase↗